The biosynthesis of terpenoids (isoprenoids) begins with the synthesis of geranyl diphosphate (GPP; C10), farnesyl diphosphate (FPP; C15) and geranylgeranyl diphosphate (GGPP; C20), which are straight chain prenyl diphosphates, through the condensation reaction of isopentenyl diphosphate (IPP; C5) with an allylic diphosphate substrate in succession (FIG. 1). In FIG. 1, the abbreviations and words in boxes represent enzymes. Specifically, hmgR represents hydroxymethylglutaryl-CoA (HMG-CoA) reductase; GGPS represents GGPP synthase; and FPS represents FPP synthase.
Among prenyl diphosphates, FPP is the most important intermediate for the biosynthesis, and is a precursor for the synthesis of numerous kinds of terpenoids, e.g., steroids including ergosterol (provitamin D2), the side chains of quinone (vitamin K; VK), sesquiterpenes, squalene (SQ), the anchor molecules of farnesylated proteins, natural rubber, etc.
GGPP is also an important intermediate for the terpenoid biosynthesis, and is essential for the biosynthesis of such compounds as retinol (vitamin A; VA), β-carotene (provitamin A), phylloquinone (vitamin K1; VK1), the anchor molecules of geranylgeranylated proteins, the side chains of chlorophyll, gibberellins, and the ether lipid of archaea.
Farnesol (FOH; C15) and geranylgeraniol (GGOH; C20), which are alcohol derivatives of FPP and GGPP, respectively, and their isomers such as nerolidol (NOH; C15) are known as fragrant substances in essential oils used in perfumes. They are also important as starting materials for the synthesis of various compounds including the above-mentioned vitamins useful as pharmacological agents (FIG. 1).
Thus, it is desired to establish a system in which a pure product of the so-called active-type prenyl alcohol, not a mixture of cis- and trans- ((Z)- and (E)-) isomers, can be produced in a large quantity.
Although it had been believed that all the biosynthesis of IPP is performed via the mevalonate pathway (the pathway in which IPP is synthesized from acetyl-CoA through mevalonate), M. Rohmer et al. elucidated a novel pathway for IPP synthesis using bacteria at the end of 1980's. This is called the non-mevalonate pathway or DXP (1-deoxyxylulose 5-phosphate) pathway, in which IPP is synthesized from glyceraldehyde-3-phosphate and pyruvate through 1-deoxyxylulose 5-phosphate.
GGOH is currently produced by chemical synthesis (see, for example, Japanese Unexamined Patent Publication No. 8-133999). However, the chemical synthesis of GGOH requires more steps than that of FOH or NOH with shorter carbon chains, and thus requires a higher cost. Besides, though chemically synthesized GGOH has the same carbon skeleton as that of naturally occurring GGOH, it is obtained as a mixture of (E)-type (trans type) and (Z)-type (cis type) in double bond pattern. (E, E, E)-GGOH (hereinafter, abbreviated to (all-E)-GGOH) is the form synthesized in metabolic pathways in organisms and is industrially valuable. In order to obtain (all-E)-GGOH in a pure form, refining by column chromatography, high precision distillation, etc. is necessary. However, it is difficult to carry out high precision distillation of GGOH that is a thermally unstable allyl alcohol. Also, refining by column chromatography is not suitable for industrial practice since it requires large quantities of solvents and column packings, as well as complicated operations of analyzing and recovering serially eluting fractions and removing the solvent; thus, this method is complicated and requires a high cost. Under circumstances, it is desired to establish a method of biosynthesis of (all-E)-GGOH by controlling the generation of (E)- and (Z)-geometrical isomers or by utilizing characteristics such as the repeat structures of reaction products. However, such a method has not been established yet. The substrates for GGOH synthesis are provided via the mevalonate pathway in cells of, for example, budding yeast Saccharomyces cerevisiae. However, even when HMG-CoA reductase that is believed to be a key enzyme for GGOH synthesis was used, the use only increased the ability of squalene synthesis through FPP synthesis (Japanese Unexamined Patent Publication No. 5-192184; Donald et al., (1997) Appl. Environ. Microbiol. 63, 3341-3344). Further, even when a squalene synthase gene-deficient strain of a special budding yeast that had acquired sterol intake ability was cultured, accumulation of 1.3 mg of FOH per liter of culture broth was only revealed (Chambon et al., (1990) Curr. Genet. 18, 41-46); no method of biosynthesis of NOH has been known. With respect to the biosynthesis of GGOH, production of 0.66-3.25 mg per liter of culture broth is achieved by culturing plant cells in Japanese Unexamined Patent Publication No. 9-238692. However, this method needs an expensive plant cell culture medium inappropriate for industrial application and also requires light for culturing cells. Thus, this method is less practical even compared to the conventional GGOH preparation from natural products such as essential oils. There is known no method of biosynthesis of GGOH suitable for industrialization, e.g., biosynthesis by culturing microorganisms.